Fluorescence is one of the physical phenomena where light is emitted by a material as a result of first absorbing light or other electromagnetic radiation. The emitted light typically has a lower energy (longer wavelength) than the light absorbed. Fluorescence is an invaluable measurement tool with applications ranging from mining to biology. In consequence, countless instruments have been constructed to measure various aspects of fluorescence from excited materials. One application of the present invention optimizes the transient recording of fluorescent emissions from biological samples as follows. LED (instead of laser) excitation is used to minimize photochemical effects (i.e. spectral hole burning) and to excite all available fluorophores by matching the absorption spectrum of the fluorophore with the broad spectrum of the LED. Fluorescent lifetime can be determined from the recorded signals. Pluralities of time constants may be indicative of a mixture of a molecule in a plurality of physical states. Information of this nature may be helpful for cancer diagnosis, forensic tissue examination, and other areas of research.
The present invention can further optimize the transient recording of fluorescent emissions by using the gating paradigm of photomultiplier tubes (PMT). The PMT is gated off during LED excitation to avoid PMT saturation allowing for higher intensity LED or laser pulses. This technique has been used abundantly in scintillation counting for high-energy physics [1], but has rarely been used for biological applications. This is because in scintillation counting, the shape of the signal is less important than the total energy and the occurrence of events, but in biology the shape of the signal (initial intensity and time constants/lifetimes) correlates with the fluorophores being excited. Gating introduces significant systematic error that is sometimes difficult to remove and can obscure intensities and lifetimes [2]. However, modern electronics and basic signal processing can greatly enhance the capabilities of these kinds of instruments by increasing speed and reducing systematic error.
Transient properties of fluorophores can also be studied by using sinusoidal excitation and observing the resulting modulations of fluorescence intensity and phase. Modulations in phase and intensity can be used to estimate the fluorescent lifetimes.
LED's can be crafted with a wide range of spectral properties and are of particular interest because their spectral range can be matched to the absorption spectrum of fluorophores. LED output intensity also responds very quickly to changes in current, but due to being semiconductor devices, the relationship between current and intensity is not very linear close to the voltage threshold.
Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.